The growth of solid tumors induces and requires a network of growth signals between the tumor cells and the surrounding tissues. The tumor cells often secret growth factors that promotes self-growth (autocrine action) and stimulate angiogenesis (paracrine action), which is the recruitment of blood vessels, to support the tumor growth in vivo (Folkman, et al., Semin. Oncol. 29: 15 (2002)). Ideally, therapeutic agents that block both autocrine and paracrine functions will be more effective in treating cancer patients.
Pleiotrophin (PTN) is a secreted, heparin-binding growth factor that is highly expressed during development but with very specific and limited expression in normal adult tissues (see review by Muramatsu, J. Bio. Chem. 132: 359-371 (2002)). PTN has been shown to stimulate the growth of fibroblasts, endothelial cells, and different tumor epithelial cells in vitro (Fang et al., J. Bio. Chem. 267: 25889-25897 (1992)).
Over-expression of PTN in cells results in malignant phenotypes that include anchorage-independent growth in vitro and tumorigenicity in nude mice (Chauhan et al., Proc. Natl. Aacd. Sci. 90: 679-682, (1993)). PTN also displays angiogenic activity in the rabbit corneal assay when it is expressed in MCF-7 cells (Choudhuri et al., Can. Res. 57: 1814-1819 (1997)). Screening of various human tumor cell lines and tumor specimens has revealed that PTN is expressed in nearly 50% of tumor cell lines of different origin, such as melanoma, neuroblastoma, breast, prostate, lung, and pancreatic cancers (Nakagawara et al., Can. Res. 55: 1792-1797 (1995); Jager et al., Int. J. Cancer 73: 537-534 (1997); Czybayko et al., Proc. Natl. Aacd. Sci. 93: 14753-14758 (1996); Vacherot et al., The Prostate 38: 126-136 (1999); Weber et al., Can. Res. 60: 5284-5288, (2000)). In addition, the PTN level in serum is elevated in pancreatic, lung and colon cancer patients; and it may correlate with disease stages (Jager et al., British J of Cancer 86: 858-863, (2002); Klomp et al., Clinical Cancer Res. 8: 823-827, (2002)). Moreover, depletion of PTN by ribozyme in tumor cells suppresses tumor growth and metastasis in mice including melanoma, (Czubayko et al., J. Bio. Chem. 269: 21358-21363, (1994); Czybayko et al., Proc. Natl. Aacd. Sci. 93: 14753-14758, (1996); Aigner et al., Gene Therapy. 9: 1700-1707 (2002)), choriocarcinoma (Schulte et al., Proc. Natl. Aacd. Sci. 93: 14579-14764 (1996)) and pancreatic cancer (Weber et al., Can. Res. 60: 5284-5288, (2000)).
U.S. Pat. No. 5,770,421 discusses the isolation and cloning of a receptor of PTN, ALK (Anaplastic Lymphoma Kinase), as well as the use of the anti-ALK antibodies for detecting ALK (This and all other U.S. patents and patent applications cited herein are hereby incorporated by reference in their entirety). Ribozyme-mediated reduction of ALK leads to the reduction of tumor size and increase of the animal survival in xenograft model of U87MG cells (WO 0196394A2 and Patent Application U.S. 2002/034768A1). Therapeutic use of antibodies against ALK has not been examined in any of the above-referenced publications.
It is therefore desirable to develop blocking reagents, such as antibodies, that block PTN functions including oncogenic and angiogenic activities.
The development of therapeutic antibodies often begins with raising antibodies in mice against an injected human protein target. It is generally difficult to elicit an adequate immune response against proteins that are highly homologous among the mammalian species because the mouse immune system cannot differentiate between the human version of the protein and its own version of the protein. PTN is nearly identical among mammalian species (see FIG. 1 for human and mouse PTN sequence comparison) and thus it is very difficult to generate antibodies against PTN in rodents, especially the neutralizing anti-PTN antibodies. One example of an anti-PTN monoclonal antibody used for the diagnostic purpose is disclosed in PCT Publication No. WO 0020869A1.
The present invention is directed to novel methods of making antibodies against any target proteins. These methods are particularly useful in producing antibodies against the proteins that are highly homologous among the mammalian species. These methods have given rise to multiple neutralizing anti-PTN monoclonal antibodies, which functionally inhibit or block the angiogenic and oncogenic activities of PTN in vitro and/or in vivo, and are therefore of great therapeutic value. The present invention also provides for effective methods of inhibiting cancer growth and angiogenesis in a subject with antagonists of PTN, preferably neutralizing anti-PTN antibodies.